Abstract Numerous medical and scientific studies indicate that autophagy plays a key role in many diseases including neurodegenerative, infective, and cardiac diseases, and cancer. Reducing or promoting autophagic flux during particular stages of these diseases can improve patient outcomes. To understand this complex relationship between these diseases and autophagy, we must first understand what dictates its timing and regulation. Successful completion of autophagy requires a host of proteins, including Atg3. Atg3 catalyzes the conjugation of Atg8 (or LC3 in mammals) to the PE lipids in the autophagic membrane. The reaction product, Atg8-PE, acts as a marker for autophagic cargo and allows the autophagosomal membrane to be constructed. Previous studies have provided some molecular insights into this reaction; however, our understanding of its mechanism has remained remarkably elusive. In addition, Atg3's function hinges on an N-terminal amphipathic helix (NAH). This helix recognizes highly curved membranes and is required for effective Atg8-PE conjugation in vivo. In this study, we plan to examine the structural and molecular basis of human ATG3 (hATG3) activation and regulation by its interaction with the membrane for this conjugation reaction. In Aim 1, we will determine the structures and dynamics of hATG3 and its thioester intermediate hATG3-LC3. Structural models derived from high-resolution NMR will be validated using in vitro conjugation and in vivo function assays. In Aims 2 and 3, we will analyze the structural and molecular basis of ATG3 activation and regulation, and determine the molecular mechanism that drives the selective binding of ATG3's NAH to strongly curved membranes, respectively. Together, these studies will provide a mechanistic insight into hATG3 activation and regulation for the production of LC3-PE conjugate, a key molecule that triggers membrane expansion and recruits cargos for formation of the autophagosome in autophagy. Our results will contribute to a fundamental understanding of the autophagy process, which may lead to the development of new disease treatments and eventually improve the clinical patient outcome.